Diagnostic agents for the detection of urobilinogen materials in body fluids

ABSTRACT

Diagnostic agents for use in the detection of urobilinogen materials in body fluids such as urine, the agents comprising at least one strong acid and at least one compound which reacts with urobilinogen materials present in the body fluids to provide analytically measurable dyestuffs wherein said compound has the formula:   IN WHICH R1 and R2 which may be the same or different, each represent halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy, straight-chained and cyclic, saturated and unsaturated secondary and tertiary amino, or R1 and R2 together represent oxygen, sulfur, sulfonyl, alkylthionium, imino, alkylimino, substituted or unsubstituted arylimino or open-chained, alicyclic or heterocyclic dialkylimino, R2 can also represent the residue of secondary straight or branched, openchained or mono- or polycyclic alkyl or aralkyl and wherein one of R1 and R2 can also represent hydrogen, R3 is hydrogen or alkyl, and n and m which may be the same or different each have a value of 0, 1, 2 or 3.

United States Patent [72] Inventors 7 Walter Rlttersdorf;

Hans-Georg Rey; Peter Rieckmann, all of Mannheim-Waldhot, Germany [2!]Appl. No. 768,882

[22] Filed Oct. 18, 1968 [45] Patented Dec. 28, 1971 [73] AssigneeBoehringer Mannheim GmbH Mannheim, Germany [32] Priorities Oct. 26, 1967[3 3 Germany July 3, 1968, Germany, No. P 17 67 931.1

[54] DIAGNOSTIC AGENTS FOR THE DETECTION OF UROBILINOGEN MATERIALS INBODY FLUIDS OTHER REFERENCES J. Fischl and N. Pinto, Clinica ChimicaActa, 2, 527- 33 (1957).

Primary ExaminerMorris O. Wolk Assistant Examiner-Sidney MarantzAttarney--Burgess, Dinklage & Sprung ABSTRACT: Diagnostic agents for usein the detection of urobilinogen materials in body fluids such as urine,the agents comprising at least one strong acid and at least one compoundwhich reacts with urobilinogen materials present in the body fluids toprovide analytically measurable dyestuffs wherein said compound has theformula:

3 in which R, and R, which may be the same or different, each representhalogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy, acyloxy,alkoxy, straight-chained and cyclic, saturated and unsaturated secondaryand tertiary amino, or R, and R together represent oxygen, sulfur,sulfonyl, alkylthionium, imino, alkylimino, substituted or unsubstitutedarylimino or open-chained, alicyclic or heterocyclic dialkylimino, R,can also represent the residue of secondary straight or branched,open-chained or monoor polycyclic alkyl or aralkyl and wherein one of R,and R, can also represent hydrogen, R is hydrogen or alkyl, and n and mwhich may be the same or different each have a value ofO, l, 2 or 3.

DIAGNOSTIC AGENTS FOR THE DETECTION OF UROBILINOGEN MATERIALS IN BODYFLUIDS The present invention relates to new and improved diagnosticagents for the detection of urobilinogen bodies in body fluids. Moreparticularly, this invention relates to improved diagnostic agents forthe detection of urobilinogen bodies in urine, spinal fluids and otherbody fluids.

It is known that urobilinogen bodies (bilans), indole, sulfonamides,porphobilinogens, urine indican and S-hydroxy-indole-acetic acid can bedetected using a solution of pdimethylaminobenzaldehyde in hydrochloricacid. This detection test is known in the literature as Ehrlichsreaction. It has achieved considerable importance, particularly inmedical diagnosis, for the detection of increased urobilinogens" in theurine. Although the test is not very specific, it has come to beregarded as the standard test method for the diagnosis of diseases ofboth the liver and gall bladder.

In an attempt to simplify this test, it has already been proposed tomake up test tablets (British Patent Specification No. 779,921) in whichthere is present pdimethylaminobenzaldehyde, and a solid inorganic ororganic acid (also U.S. Pat. No. 2,320,282 directed to test tablets forthe detection of sulfonamides). When such tablets are dissolved in moreor less diluted urine, there is obtained a color reaction whichcorresponds approximately to that produced in accordance with theoriginal Ehrlich test. However, for the evaluation of the color reactionproduced when, for example, sulfonamides are present, it is necessary toadd a blue auxiliary dyestuff, such as methylene blue.

In accordance with a further proposal for the simplification of theEhrlich test, the reaction has also been carried out employing testpapers, (Fischl and Pinto, Clinica Chim. Acta, 2,

527533/, and French Patent No. l,450,273, in which the phosphoric acidused by Fischi and Pinto is replaced by oxalic acid).

It is an object of the present invention to provide an improveddiagnostic agent for use in the detection of unrobilinogens present inbody fluids which avoids the disadvantages of the art.

Another object of the present invention is a simple and practicaldiagnostic agent for use in the detection of urobilinogens present inbody fluids comprising a-test paper or film.

Other objects will appear hereinafter.

In accordance with the invention, it has now been found that diagnosticagents for use in the detection of urobilinogens present in body fluidsare obtained by employing a strong acid in admixture with a derivativeof p-aminobenzaldehyde.

Preferred p-aminobenzaldehyde derivatives to be used according to thepresent invention are those which carry on the p-amino group, a branchedalkyl group or a substituent which possesses strong electron attractingproperties. Illustrative of the compounds of this type are those havingthe formula:

wherein R, and R,, which may be the same or different, are each halogen,cyano, nitroso, carbalkoxy, carbamido, hydroxyl, acyloxy, alkoxy oropen-chained or cyclic, saturated or un saturated, secondary or tertiaryamino or R and R together represent oxygen, sulfur, sulfonyl,alkylthionium, imino, alkylimino, unsubstituted or substituted aryliminoopenchained, alicyclic or heterocyclic dialkylimino radical, R, can alsobe the residue of a secondary straight-chained or branched,open-chained, monoor polycyclic alkyl or aralkyl and wherein one of thesubstituents R, and R; can be hydrogen, R represents hydrogen or alkyland n and m, which may be the same or different, are 0, l, 2 or 3.

In preparing the diagnostic agents according to the invention, thecompounds of the above formula (I) are preferably applied to anabsorbent carrier, together with a strong, solid acid. The compounds (I)can also be used for the preparation of test film strips according tothe procedure described in published Dutch Pat. application No. 67.15828 and also in a form suitable for the detection of urobilinogens insolutions.

The preferred embodiment of the new diagnostic agents according to thepresent invention, i.e., test paper strips are prepared by impregnatingan absorbent carrier, preferably filter paper, with a solution whichcontains 0.00 [-1 percent of an aldehyde of formula I and at least 3percent, preferably 15-30 percent, of a strong, solid acid.

Examples of suitable strong, solid acids include sulfosalicyclic acid,p-toluene-sulfonic acid, potassium bisulfate, glutamic acidhydrochloride and preferably oxalic acid.

The impregnation solution can also contain inert additives, such as forinstance polyvinyl alcohol, polyvinyl pyrrolidone, copolymers ofpolyvinyl pyrrolidone and polyvinyl acetate, polyglycols and the like.

As solvents for use in preparing the solution for use in theimpregnation of the absorbent carrier, there are suitable all readilyvolatile solvents in which the aldehydes (I) and also the acids aresufficiently soluble. Consequently, it is preferred to use polarsolvents, such as methanol.

After impregnation, the filter papers are dried, out up and, if desired,sealed on the synthetic resin foils or sealed between synthetic resinfoils. When rodlets or strips are used as absorbent carriers then theyare ready for use immediately after drying.

In carrying out the test reaction according to the present invention forthe detection of urobilinogens or of unrobilinogen bodies, the newdiagnostic agents according to the present invention are dipped into thesolution to be investigated and the color change then evaluated after ashort period of time, i.e., l to 2 minutes.

In contradistinction to the previously known diagnostic agents availablefor the detection of urobilinogens, the new diagnostic reagentsaccording to the present invention are substantially more sensitive andare capable of detecting amounts as low as about 0.4 mg.-percenturobilinogen in urme.

Surprisingly, this color reaction is not disturbed by the presence ofurea. Even a 3percent solution of urea hardly causes any color change totake place on the new test papers according to the present invention,whereas test papers which contain p-dimethylaminobenzaldehyde take on anintense yel' low color.

Furthermore, it could not have been foreseen that the new diagnosticagents according to the present invention would not be adverselyaffected either by urine indican or by indole or by sulfonamides (in thecase of normal dosages) in urine. This finding is particularlysurprising because the compounds (I), dissolved in 2-20 percenthydrochloric acid, can be used very satisfactorily for the detection ofindole in solution. For the detection of urobilinogens and ofurobilinogen bodies in solutions, p-dimethylaminobenzaldehyde can, ofcourse, be replaced by compounds of formula I and the content thusdetermined photometrically in a cuvette in a very exact manner and freeof any interference.

A preferred group of benzaldehyde derivatives according to the presentinvention are compounds having the formula:

H (CH) R:

chained or monoor polycyclic alkyl or aralkyl radical, R is hydrogen oralkyl and nis 0, 1,2 or 3.

Thus according to one aspect of the present invention, there is provideda diagnostic agent for the detection of urooxalic acid 200 g. polyvinylalcohol 3.0 water 10.0 ml.

methanol uil I001) ml.

bilinog cn b i l i Particularly in i f 5 Following drying of theimpregnated paper there was obco"1pmes an absorbfmt 9 3 a Strong solldtained a white test paper having the same properties as that and asubstance which, in the manner of the Ehrlich test, produced according[0 example 1 produces a color change in the presence of urobilinogenbodies, characterized in that as color-producing substance 10 EXAMPLE3there is used at least one compound of formula I.

. F According to a further aspect of the present invention, there a ssil s rz fi iz gllgfifg 1x1; i is provided a process for the detectionof urobilinogen bodies 8 p p e var:-

. ous concentrated solutions containing x parts of p-N-(N present inbody fluids, the process being carried out throughmethyl-piperazino)-benzaldehyde (MPBA) or of pthe use of a test strip,i.e., an absorbent carrier or, a reagent l5 dimethylaminobenzaldehyde(DABA) and parts of oxalic film or in solution in the presence of astrong, solid or liquid acid in 100 parts of methanol. All of the testpapers thusly obacid and a substance which, in the manner of Ehrlich stest, i

. tained were white following drying. The dried test papers wereproduces a color change in the presence of urobilinogen dipped eitherinto a 3 percent solution of urea or into a urine bodies, characterizedin that a compound of formula I is used sample which was free ofurobilinogen. The color changes as the color indicator. 20

which resulted are set out in the following table: For a fullerunderstanding of the nature and ob ects of this invention, reference maybe had to the following exari rles,

. TABLE I which are given merely as further illustrations of theinvention and are not to be construed in a limiting sense. x MP DAMEXAMPLE 1 0.05 white yellow Filter paper (Schleicher & Schiill No. 23l6)was im- 0. 75 whi e yell w t 0.l white yellow pregnated with a solutionhaving the following composition. 01 pal: only yellowish yellowpbis-(fi-dicthylaminoethyl)-aminohenzaldehyde 0.2 g. oxalic acid 20.0 g.

h l d l00.0 l. am a m As can be clearly seen from the above results, thetest paper Following drying of the impregnated paper there was accordingto the present ltlVetlllOl'l, in contradistinction to the known testpaper, is substantially insensitive to urea and thus tamed a white testpaper which, with normal urine, gave a pale k l r d h t d reacts moresensitively and more specifically with urof 8 an M g giF F con l "59bilinogen bodies as the pink color reaction with urobilinogen anmg"perccfn f i i ms gave j mg is not masked by the more or less stronglyyellow color reacfi 0 T I "T t F or tion with urea. It is, of course,obvious that such a yellow color swePm to no et Co Urme Specimens me oum "f 40 reaction considerably impairs the sensitivity and measurabilitybodies, as well as 3 percent aqueous solutions of urea, .dldnot oftheurobilinogen reaction bring about any color change in the test paper.Urine indican also did not produce a color reaction. The test paperscould be EXAMPLE 4 evaluated after 1-2 minutes followin wettin with theli uid g E q Flltet paper (Schleicher & Schull No. 235 or 2316) was1111- samples to be tested.

pregnated with methanolic solutions which, per I00 parts of EXAMPLE 2solution, contained A parts of the compounds I as given in the h nfollowing table and B parts of oxalic acid. White test papers Filterpaper (Schleicher & Schull No. 23 l6) was imwere thereby obtained ineach case Pregnated 3 Soluuo" having the following composltloni Thecolor reactions which resulted following immersion of these test papersinto various solutions were evaluated after p- -tfly y ly gl-2 minutes.The results are set out in table ll which follows:

TABLE II Color reaction with- Urobllin ogen-free Urobiliri ogen-Compound (I) A B urine=3% urea soln. Normal urine containing urinep-N,N-dimethy1-N'-ethy1hydrazino-benzaldehyde 0.1 25 YeiiowishYellow-pink... Bed-violet. p-N-riitrosoN-methylaminobenzaldehyde 0.1 dYel1owish. Orange. p-N-cyanomethyl-N-methylamirio-benzalde 0.1 Pale pinPink-red. p-bis-(carbethoxymethyl)aminobenzaldehyde. 0.1 .do D0.p-N-B-chloroethyi-N-methy1aminobenzaldehydo 0.1 Yellow-pink. Purple.p-N-Bhydmxyethyl-N-inethylaminobenzaldehyde 0. 1 do 1 D0.p-N-fi-metlioxyethyl-N-methylsmiiiobenzaldehyde O. 1 .do. Do.p-bis-(B-fluoroethyD-amino-benzaldehyde. 0.1 Pale pink- Blue-red s(B-chloroethyl)-imiino-benza1dehyde. 0. 1 d0. D0. p(B-bromoethyl)aminobenzaldehyde. O. 1 do Do.p-bis(fl-hydroxyetliy1)-aminobeiizaiehyde 0.1 Yellow-pink Purple.p-bis(B-methoxyethyl)-amlno-benzaldehyde 0. 1 -do D0.p-bis-(B-cyanoethyl)aminwbenzaldehyde .s 0.15 Pale pink Pink-redp-bis-(B-dimethylaminoethyl)-aminobenzaldehy 0. 2 -do s Do. p-bis(fl-diethylaminoethyi)-arriinobenzaldehyde. 0. 2 do D0.p-bis-(B-N-piperidinoethyl)-amlnobenza1dehyde 0. 1 .do Do. [p-bis-(fi-triethylammoriiumethyl) aminobenzaldehy 0. 2 .do D o.[p-bis-(N-pyridinlumethyl)-aminobenzaldehyde]dibromide 0.2 .do Do.p-N-inorpholino-benzaldehyde 0.1 Yellow-pinto Red-violet.p-N-tliiomorpholino-benzaldehyde. 0. 1 ..do Do.[p-N-(S-methylthio-morpholinium)- 0.2 Pale pink Plnk'rcd.p-N-(S-dioxothio-morpholino)-benzaldehyde 0. 1 .do. D0.[p-bis-(N-quinolinium-ethyl)amino-benzaldehyde] dibroruid 0.2 do D0.p-N-piperazlnobenzaldehyde 0. 1 .dO D0-p-N-(N-methylpiperazino)-benzaldehyde. 0. 05 .do Do.p-N-[N-(p-formyl-pheiiyl)-piperazino]-benzald y 0.1 2 y .do...Red-violet. [p;N-(l1{-dirnethyl ipgazin r il be zmil h d Boiling. .Q-.Qs)flilLQ--- .m 19811,"...

Compound (I) Lp-N-(N'-cyc1epentamethylenepiperazinium) -benzaldehyde-iodlno- A U p-N-[Ncyclo-(3-oxapentamethy1ene) -pigergzinium1-b enzalehydo iodinepgis-carbethoxy-ethyl)-amino-benzalde y pi5-p-bis-(y-bromopropyl)-a.minobenzaldehydep-bis-(v-eyanopropyl)-aminobenzaldehyde F-bis-(v-dimethyl-aminopropyl)-aminbenza1dehyde p-bis-('y-trimethybampnoniumpropyl)-aminobenzaldehyde]dichloride. p-Ndethylamino-ethyl-N-methylaminobenzaldehydep-N-d1methylamino-ethyl-N-meth laminobenzaldehyde.p-N-diisopropylaminoethyl-N-met ylaminobenzaldehyde...p-N-dlethylaminoethyl-N'othylaminobenzaldehydep-(l-methyl-l,5-diazacyclooctyl-5)-benzaldehydep-N-cyanoethyl-N-methylaminobenzaldehydep-N-cyanoethyl-N-ethylaminobenzaldehyde.p-N-v-dimethylaminopropyl-N-methyl-aminobe aidep-N-bis-sulioethylaminobenzaldehydep-N-diethylamino-ethyl-N-isopropylamino-benzaldehyp-N-cyanoethyl-aminobenzaldehyde carbamidoethyl)-amlnobenzaldehyde.l

TABLE I! :Cunlinucd Color reaction with Urobilin ogeniree Urobilin ogen-A B urine=3% urea soln. Normal urine containing urine 0.2 do do Do. 0.220 do. do l)o. 0.1 20 Yellowisl Yollowishplnk Red. 0.1 20 Yellowishnh.Yellow-pink... ltcd.

0.05 20 Pole-yellowish. Pnlo pink Pink-rod. 0.05 20 do. 0.05 20 d0. 0.0520 White 0.05 Pale yellowish 0.05 25 ..do

0.05 25 d0 0.05 25 ....do. I) 0.05 25 do 0.05 25 Yellowish. ..do I'll0.05 25 do Ycllcwish pink. 0.05 25 Yellow Yellow-plnk. llo. 0.05 25Yellowisl Polo pink Pink-rod. 0.06 25 do... do m'p 0.01 20 White do..Pink-roll 0.05 20 Pale yellowish do Do.

p-N-diethylamino-ethylamjnobenzaldehydeI:1:11:.

5 Strips offilter paper (Schleicher & Sch'rill No. 2316) wereimpregnated either with a solution of 0.05 g.p-N-(N-methylpiperazino)-benzaldehyde (MPBA) or ofpdimethylaminobenzaldehyde (DABA) and 20 g. oxalic acid in 100 ml.methanol and then dried.

The dried papers were then immersed into aqueous solutions ofconventionally used sulfonamides and evaluated after 1-2 minutes. Thecolor changes which were observed are set out in tablel l l whichfqllows: A

TABLE HI No coloration was observed when urine free of urobilinogen wastested in the same manner.

EXAMPLE 7 25 ml. urine were acidified with 12 ml. glacial acetic acidand extracted with 40 ml. ether. The extract was washed twice with 20ml. amounts of water and again made up to 40 ml. with ether. 20 ml. ofthis extract was mixed with 0.5 ml. of a solution of l g.p-N-morpholino-benzaldehyde in 5 ml. concentrated hydrochloric acid andthe resultant mixture shaken vigorously. Thereafter, there were added 6ml. of a semisatu- N-(3,4-dimethyl-5-isoxazolyl)-sulfanila- EXAMPLE 6For use in the production of a reagent film suitable for the detectionof urobilinogens, there was prepared a mixture of the followingcomponents:

p-bis-(Bcyanoethyl)-aminobenzaldehyde 0.75 g. syrupy orthophosphcricacid 5.00 g. colloidal silicic acid (Aerosil) 6.00 g. organic sodiumsulfonate (rapid wetting agent) 2.00 g. polyvinyl butyracetal (Mowital")l0.00 g. polyethylene glycol 6,000 [0.00 g. methanol l00.0 ml.

There was applied a 300 othick layer of the above mixture onto a whitepolyvinylchloride foil, followed by drying with warm air. Awater-insoluble film was thereby formed. When this was moistened with adrop of urobilinogen-containing urine, after l-2 minutes had elapsedthere was observed a red color which, following wiping off the urine,became darker.

rated solution of sodium acetate, followed by shaking up again. The redmaterial which was formed thereby collected in the aqueous phase. Afterseparation, the ether phase was again washed with 2 ml. water. Thecombined aqueous extracts were made up to 20 ml., the color presentmeasured in a photometer at 557 nm. and the result evaluated by means ofa previously prepared calibration curve.

EXMZP 8 Filter paper (Schleicher 81. Sclii'rll No. 23i6) was impregnatedwith a solution of 0.5 parts pbis-(diethylaminoethyl)-aminobenzaldehydebis-hydrogen oxalate, 30 parts maleic acid and 0.5 parts polyethyleneglycol 1,000 in parts methanol, and then dried.

Strips of this paper reacted with urobilinogen-containing urine toprovide a pink to red color.

Papers with the same analytical properties but which were easier tounroll and cut up were obtained by additionally using 5 partspolyethylene glycol 6,000 or 20,000.

EXAMPLE 9 Filter paper (Schleicher & Scliiill No. 2316) was impregnatedwith a solution having the following composition:

p-(cyelohexylamino)-benzaldehyde 0.05 g. oxalic acid 20.0 g. methanol ad100.0 ml.

After the impregnated paper was dried there was obtained a white testpaper which, when moistened with urine samples containing urobilinogenbodies, gave a more or less intensive pink-to-violet colorationdepending on the urobilinogen concentration. Urine samples having a verylow content of urobilinogen bodies and 3 percent aqueous solutions ofurea did not give any color change in the test paper. Urine indican alsogave no color reaction. The test papers can be evaluated for colorchange after 1-2 minutes.

EXAMPLE Filter paper (Schleicher & Schr'ill No. 23l6) was impregnatedwith a solution having the following composition:

p'Uec.-butylnmlno)-benzaldehyde 0.05 g. potuulum hllulfute l5.0 g.polyethylene glycol 5.0 3. water ad l0l).0 ml.

After drying the impregnated paper, there was obtained a white testpaper which had the same properties as the test paper produced accordingto example 9.

EXAMPLE 1 l Strips of filter paper (Schleicher & Schiill No. 2316) wereimpregnated with solutions having various concentrations containing Xparts p-(isopropylamino)-benzaldehyde (iPABA),p-(n-propylamino)-benzaldehyde (nPABA) or'p-(dimethylamino)-benzaldehyde (DABA) and parts oxalic acid in 100 partsmethanol, according to the procedure described in Example 1. The driedtest papers were dipped into 3% solutions of urea. The color changeswhich were thereby observed are set out in the following Table:

As can be clearly seen from the results set out in the above table, thetest papers according to the present invention which containedp-(isopropylamino)-benzaldehyde were substantially less sensitive tourea and thus reacted more sensitively and specifically withurobilinogen bodies as the pink color reaction with urobilinogen was notmasked by the more or less strong yellow color reaction with urea.

EXAMPLE l2 TABLE V Reaction with Reaction 3% urea soln. or with urowithurine of bilinogenlow urobilinogen containing Compound (1) content urinep-(lsopropylamlno)-benzaldehyde White Pink-red.11-(isopropylmnino)-m-mnthyl Pale yellowish" Do.

Imuznk ohydo. i-(suwhuLylnmino)-lmnzuldullyrlu .do Do.p-(mntyl-(immlno)-bunzuldullydu Yellowisln. Do. i-(2,4-ilmothylpnntyli-muluo)- Yellow Rod.

hunzuldoliydo. p-(cyclopentylamlno)-bcnznldehyde Polo ed- V yellowish.yiolet TABLE V..C0niinucd Reaction with Reaction 3% urea soln. or withurowith urine of biilnogeulow urobilinogen containing Compound (I)content urine p-(it-mathyl-cyclopentylamino)- Yellowish Do.

benzaldehyde. p-(cyclohexylamino)-benzaldehydo Almost white Do.p(cyclohexylamino)-m-methyl- Pale Do.

benzaldehyde. yellowish. p-(cyclohexylamino)-m-isopropyl- Ycllowish Do.

benzuldehydo. p-(2-mothyl-cyclolwxylanilno)- .rlo Do.

bouzaldohydo. p-mouthylanil:iolmnznldullydu Yullow mung.p-(l-uy0lohuxyl-pro iyl-2-iuuino)- Yollowlshvw l'luk-I'ml.

hunroldnliyrln. p-(cyelndorlecylnmllm)-lmnzulriohyxlo.. l'nlu l'lnk.

yellowish. p-(norboruyl-Z-mnino)-beuzuldehydo Yellow Pink-rod.p-(a-mothyl-bsnzylamino) Yellowlsh Do.

benzaldehyde. p-(a-iso r0 ylbenzylamino)- do.

home do yde. p-(l-phenyl-propyl-Z-amino)- do.

benzaldchyde. p-(indanyl-Z-omino)-benza1dehyde ..do

p-(N-diethylarninoethyl-N-isopropyldo..

amino) -benzaldehyde.

strips dippe d into urine had a weak yell ow coloration due to thcinherent color ol'the urinc.

EXAMPLE iii For the production of a filmlike reagent layer for use inthe detection of urobilinogens, there was prepared a mixture of thefollowing components:

p-(cyelohexylamino)-bcnzaldehydc 0.05 g. colloidal llllClC acid(Aerosil") L00 g. oxalic acid I000 g. polyvinyl butyracetal ("Mowit-al)0.5 g. polyethylene glycol 6000 0.5 g. methanol 30.0 ml.

The above mixture was then applied at a thickness of about 1 mm. to awhite polyvinylchloride foil and thereafter allowed to dry in warm air.A water-insoluble layer was thereby formed. When this was moistened witha drop of urobilinogencontaining urine then, following the passing ofl-2 minutes, there was observed a red coloration. No coloration wasobserved with a urobilinogen-free urine.

EXAMPLE 14 25 ml. urine were acidified with 12 ml. glacial acetic acidand extracted with 40 ml. ether. The extract was washed twice with 20ml. amounts of water and again made up to 40 ml. with ether. 20 ml. ofthe thusly obtained extract were mixed with 0.5 ml. of a solution of lg. p-(cyclopentyl-amino)- benzaldehyde in 5 ml. concentratedhydrochloric acid and the mixture shaken vigorously. Thereafter, therewere added 2.5 ml of a semisaturated solution of sodium acetate and 3.5ml. water, following which the mixture was again shaken, the red coloredmaterial thereby formed collected in the aqueous phase. Afterseparation, the ether was again washed with 2 ml. water. The combinedaqueous extracts were made up to 20 ml. and the color thereof measuredin a photometer at 557 nm. The reading obtained was evaluated by meansof a previously prepared calibration curve.

it is a general advantage of those substances l which possess branchedalkyl substituents at the amino group, that they do not react withunknown ingredients of certain urine samples. Further, it could not havebeen foreseen, that the more basic derivatives of p-aminobenzaldehydehaving a branched alkyl substituent R, at the amino group would notreact with urea, although up until now it had been found that more basicaldehydes do react better with urea.

We claim:

1. Diagnostic agent for the detection of urobilinogen bodies in bodyfluids comprising at least one strong acid and at least one compoundwhich reacts with urobilinogen bodies to prowherein A. R and R are eachmembers selected from the group consisting of halogen, cyano, nitroso,carbalkoxy, carbamido, hydroxy, acyloxy, alkoxy and amine radicalshaving at least one aliphatic hydrocarbon substituent and, if there aretwo aliphatic hydrocarbon substituents they may, together with the aminenitrogen, represent an N- aliphatic heterocycle and wherein one of R andR can represent hydrogen.

B. R and R together can represent a member selected from the groupconsisting of oxygen, sulfur, sulfonyl, alkylthionium, imino,alkylimino, substituted or unsubstituted arylimino, open-chaineddialkylimino, cyclic dialkylimino and heterocyclic dialkylimino;

C. R-(Cl-l can also represent a member selected from the groupconsisting of secondary straight-chained alkyl, branched-chained alkyl,monocyclic alkyl, polycyclic alkyl and aralkyl with the proviso that inthis case, R, can represent hydrogen only if n is R is a member selectedfrom the group consisting of hydrogen and alkyl; and

n and m are each one of 0, l, 2 and 3; and a carrier therefor.

2. Diagnostic agent according to claim 1 wherein said compound whichreacts with urobilinogen bodies to provide an analytically measurabledyestuff has the formula:

wherein R represents a member selected from the group consisting ofhydrogen, halogen, cyano, nitroso, carbalkoxy, carbamido, hydroxy,acyloxy, alkoxy and amine radicals having at least one aliphatic,hydrocarbon substituent and, if there are two aliphatic hydrocarbonsubstituents they may, together with the amine nitrogen, represent anN-aliphatic heterocycle, R represents a member selected from the groupconsisting of secondary straight-chained alkyl, branched-chained alkyl,monocyclic alkyl, polycyclic alkyl and aralkyl, R is a member selectedfrom the group consisting of hydrogen and alkyl and n is 0, l, 2 and 3,with the proviso that R. is hydrogen only when n is 0.

3. Diagnostic agent according to claim 1 wherein said carrior is a papersheet having a wettable surface impregnated with a solution containing0.001-1 percent of a compound (I) as defined in claim 1 and 15-30percent of a strong solid acid.

4. Diagnostic agent according to claim 1 wherein said strong .acid is amember selected from the group consisting ofsulp-bis-(B-diethylaminoethyll-uminobenzaldehyde 0.2 g. oxalic acid 20.0g. methanol ad 100.0 ml.

6. Diagnostic agent according to claim 1 wherein said carrier is a papersheet having a wettable surface impregnated with a solution having thefollowing composition:

p-bis-(fl-cyanoethyl)-aminobenzaldchyde oxalic acid polyvinyl alcohol3.0 g. water 30.0 ml. methanol ad l00.0 ml. c

7. Diagnostic agent according to claim I wherein said carrier is a papersheet having a wettable surface impregnated with a solution having thefollowing composition: 0.05 and 0.2 parts of a member selected from thegroup consisting of p-N (N-methyl-piperazino)-benzaldehyde andpdimethylaminobenzaldehyde and 20 parts of oxalic acid in parts ofmethanol.

8. Diagnostic agent according to claim 1 wherein said carrier is apolyvinylchloride film having applied thereon a layer of the followingcomposition:

p-bis-(fi-cyanoethyl)-aminobenzaldehyde 0.75 g. syrupy orthophosphoricacid 5.00 g. colloidal silicic acid (Aerosil") 6.00 g. organic sodiumsulfonate (rapid wetting agent) 2.00 g. polyvinyl butyracetal (Mowital")l0.00 g. polyethylene glycol 6000 l0.00 g. methanol 100.00 ml.

9. Diagnostic agent according to claim 1 wherein said carrier is aliquid containing p-N-morpholinobenzaldehyde in solution in concentratedhydrochloric acid.

10. Diagnostic agent according to claim 1 wherein said carrier is apaper sheet having a wettable surface impregnated with a solution havingthe following composition: 0.5 partspbis-(diethylaminoethyl)-aminobenzaldehyde bis-hydrogen oxalate, 30parts maleic acid and 0.5 parts polyethylene glycol l,00020,000 in l00parts methanol.

11. Diagnostic agent according to claim 1 wherein said carrier is apaper sheet having a wettable surface impregnated with a solution havingthe following composition:

p-(cyclohexylaminol-benzaldehyde 0.05 g. oxalic acid 20.0 g. methanol adl00.0 ml.

12. Diagnostic agent according to claim 1 wherein said carrier is apaper sheet having a wettable surface impregnated with a solution havingthe following composition:

p-(sec-butylamino)-benzaldehyde 0.05 g. potassium bilult'ale I50 3.polyethylene glycol 5.0 water ad l00.0 ml.

13. Diagnostic agent according to claim 1 wherein said carrier is apolyvinylchloride film having applied thereon a layer of the followingcomposition:

c p-(cyclohexylaminol-benzaldehyde 0.05 g. colloidal silicic acid(Aero|il") 1.00 g. oxalic acid l0.00 g. polyvinyl butyracetal (Mowital)0.5 g. polyethylene glycol 6000 0.5 g.

14. Diagnostic agent as claimed in claim 1 wherein said compound has theformula wherein R is a secondary alkyl group which can bestraightchained, branched or cyclic and wherein R is a member selectedfrom the group consisting of hydrogen and alkyl.

15. Diagnostic agent according to claim I wherein said carrier is apaper sheet having a wettable surface.

16. A method for carrying out an analytical test for the presence ofurobilinogen bodies in biological fluids which comprises applying to thewettable surface of a diagnostic agent according to claim 15 a smallamount of the fluid to be tested and examining said paper for visualevidence of the presence of said urobilinogen bodies.

17. Diagnostic agent according to claim 1 wherein said carrier is afoil.

18. A method for carrying out an analytical test for the presence ofurobilinogen bodies in biological fluids which comprises applying to thecoated surface ofa diagnostic agent according to claim 17 a small amountof the fluid to be tested and examining said coating for visual evidenceof the presence of said urobilinogen bodies.

19. Diagnostic agent according to claim I wherein said carrier is aliquid. open-chajned, alicyclic or heterocyclic dialkt54 20. A methodfor carrying out an analytical test for the presence of urobilinogenbodies in biological fluids which comprises admixing a diagnostic agentaccording to claim 19 with a small amount of an ether extract ofthet'luid to be tested, adding a semisaturated solution of sodiumacetate and water and examining the resultant aqueous phase for visualevidence of the presence of urobilinogen bodies.

21. Diagnostic agent according to claim 1 wherein said carrier is apaper sheet having a wettable surface impregnated with a solutioncontaining 0.00] to 1 percent of a compound (I) as defined in claim 1and at least 3 percent ofa strong solid acid.

22. Diagnostic agent according to claim 21 additionally containing atleast one member selected from the group consisting of polyvinylalcohol, polyvinyl pyrrolidone, copolymers of polyvinyl pyrrolidone andpolyvinyl acetate and polyglycols.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,630,680 Dated. December 28 1971 lnventofls) Walter Rittersdorf et a1.

It is certifiedthat error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

F001. 1 line '22" v "beblet," should be --tablets-' Col. 1, line 34 I va Afit r "533/" in sert -1957. 1

Col. 1, line 38 "unro-"' should be --uro- Col. 2 line 32 "unrobilinogen"should be --urobi1inog'en-- C01. 3, line 2 "ni s should be i1 is-- Col.5, line 20 Before line 21, insert --EXAMPLE 5-'--' line 21, c lelete "5"(:01 r 6 ine 53 "pbis" should be --p 'bis-- Col. 10, Claim 13 I 1st lineof composition ingredients, delete "c" before "p" FORM PO-1050 (10-69)USCOMM-DC oosvs-pes W 0.5. GOVERNMENT PRINTING OFFICE I959 036fi-33MvUNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,680.Dated December 28 1971 lfiventofls) 'Walter Rittersdorf et al'. Page 2 5v It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Column 10 Claim 13 add to the ingredient listing as a 6th line methanol30.0 ml. Column 11, Claim 19 should read. 19. Diagnostic agent accordingto claim '1 wherein said carrier is a liquid.

Signed and sealed this 7th day of November 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissionerof Patents- USCOMM-DC 6037'5-P69 U.S. GOVERNMENT PRINTING OFFICE: 1969O366-33A,

LFORM PO-IOSO (10-69)

2. Diagnostic agent according to claim 1 wherein said compound whichreacts with urobilinogen bodies to provide an analytically measurabledyestuff has the formula: wherein R1 represents a member selected fromthe group consisting of hydrogen, halogen, cyano, nitroso, carbalkoxy,carbamido, hydroxy, acyloxy, alkoxy and amine radicals having at leastone aliphatic hydrocarbon substituent and, if there are two aliphatichydrocarbon substituents they may, together with the amine nitrogen,represent an N-aliphatic heterocycle, R2 represents a member selectedfrom the group consisting of secondary straight-chained alkyl,branched-chained alkyl, monocyclic alkyl, polycyclic alkyl and aralkyl,R3 is a member selected from the group consisting of hydrogen and alkyland n is 0, 1, 2 and 3, with the proviso that R1 is hydrogen only when nis
 0. 3. Diagnostic agent according to claim 1 wherein said carrier is apaper sheet having a wettable surface impregnated with a solutioncontaining 0.001-1 percent of a compound (I) as defined in claim 1 and15-30 percent of a strong solid acid.
 4. Diagnostic agent according toclaim 1 wherein said strong acid is a member selected from the groupconsisting of sulfosalicyclic acid, p-toluene-sulfonic acid, sulfuricacid, glutamic acid, o-phosphoric acid, oxalic acid, hydrochloric acidand maleic acid.
 5. Diagnostic agent according to claim 1 wherein saidcarrier is a paper sheet having a wettable surface impregnated with asolution having the following composition: p-bis-( Beta-diethylaminoethyl)-aminobenzaldehyde 0.2 g. oxalic acid 20.0 g.methanol ad 100.0 ml.
 6. Diagnostic agent according to claim 1 whereinsaid carrier is a paper sheet having a wettable surface impregnated witha solution having the following composition: p-bis-( Beta-cyanoethyl)-aminobenzaldehyde 0.05 g. oxalic acid 20.0 g. polyvinylalcohol 3.0 g. water 30.0 ml. methanol ad 100.0 ml. c
 7. Diagnosticagent according to claim 1 wherein said carrier is a paper sheet havinga wettable surface impregnated with a solution having the followingcomposition: 0.05 and 0.2 parts of a member selected from the groupconsisting of p-N-(N''-methyl-piperazino)-benzaldehyde andp-dimethylaminobenzaldehyde and 20 parts of oxalic acid in 100 parts ofmethanol.
 8. Diagnostic agent according to claim 1 wherein said carrieris a polyvinylchloride film having applied thereon a layer of thefollowing composition: p-bis-( Beta -cyanoethyl)-aminobenzaldehyde 0.75g. syrupy orthophosphoric acid 5.00 g. colloidal silicic acid(''''Aerosil'''') 6.00 g. organic sodium sulfonate (rapid wetting agent)2.00 g. polyvinyl butyracetal (''''Mowital'''') 10.00 g. polyethyleneglycol 6000 10.00 g. methanol 100.00 ml.
 9. Diagnostic agent accordingto claim 1 wherein said carrier is a liquid containingp-N-morpholinobenzaldehyde in solution in concentrated hydrochloricacid.
 10. Diagnostic agent according to claim 1 wherein said carrier isa paper sheet having a wettable surface impregnated with a solutionhaving the following composition: 0.5 partsp-bis-(diethylaminoethyl)-aminobenzaldehyde bis-hydrogen oxalate, 30parts maleic acid and 0.5 parts polyethylene glycol 1,000-20,000 in 100parts methanol.
 11. Diagnostic agent according to claim 1 wherein saidcarrier is a paper sheet having a wettable surface impregnated with asolution having the following composition:p-(cyclohexylamino)-benzaldehyde 0.05 g. oxalic acid 20.0 g. methanol ad100.0 ml.
 12. Diagnostic agent according to claim 1 wherein said carrieris a paper sheet having a wettable surface impregnated with a solutionhaving the following composition: p-(sec.-butylamino)-benzaldehyde 0.05g. potassium bisulfate 15.0 g. polyethylene glycol 5.0 g. water ad 100.0ml.
 13. Diagnostic agent according to claim 1 wherein said carrier is apolyvinylchloride film having applied thereon a layer of the followingcomposition: c p-(cyclohexylamino)-benzaldehyde 0.05 g. colloidalsilicic acid (''''Aerosil'''') 1.00 g. oxalic acid 10.00 g. polyvinylbutyracetal (''''Mowital'''') 0.5 g. polyethylene glycol 6000 0.5 g. 14.Diagnostic agent as claimed in claim 1 wherein said compound has theformula wherein R2 is a secondary alkyl group which can bestraight-chained, branched or cyclic and wherein R3 is a member selectedfrom the group consisting of hydrogen and alkyl.
 15. Diagnostic agentaccording to claim 1 wherein said carrier is a paper sheet having awettable surface.
 16. A method for carrying out an analytical test forthe presence of urobilinogen bodies in biological fluids which comprisesapplying to the wettable surface of a diagnostic agent according toclaim 15 a small amount of the fluid to be tested and examining saidpaper for visual evidence of the presence of said urobilinogen bodies.17. Diagnostic agent according to claim 1 wherein said carrier is afoil.
 18. A method for carrying out an analytical test for the presenceof urobilinogen bodies in biological fluids which comprises applying tothe coated surface of a diagnostic agent according to claim 17 a smallamount of the fluid to be tested and examining said coating for visualevidence of the presence of said urobilinogen bodies.
 19. Diagnosticagent according to claim 1 wherein said carrier is a liquid.open-chajned, alicyclic or heterocyclic dialkt54
 20. A method forcarrying out an analytical test for the presence of urobilinogen bodiesin biological fluids which comprises admixing a diagnostic agentaccording to claim 19 with a small amount of an ether extract of thefluid to be tested, adding a semisaturated solution of sodium acetateand water and examining the resultant aqueous phase for visual evidenceof the presence Of urobilinogen bodies.
 21. Diagnostic agent accordingto claim 1 wherein said carrier is a paper sheet having a wettablesurface impregnated with a solution containing 0.001 to 1 percent of acompound (I) as defined in claim 1 and at least 3 percent of a strongsolid acid.
 22. Diagnostic agent according to claim 21 additionallycontaining at least one member selected from the group consisting ofpolyvinyl alcohol, polyvinyl pyrrolidone, copolymers of polyvinylpyrrolidone and polyvinyl acetate and polyglycols.